The major objective of this proposal is to understand the parameters of B-cell tolerance, and to apply this knowledge to the regulation of undesirable B-cell responses, specifically those that occur in AIDS. For example, early humoral responses in HIV-infected patients may be non- protective, facilitate viral entry into mononuclear cells or could sensitize bystander T cells for apoptosis by crosslinking of CD4. We propose that elimination of these humoral responses via the induction of B-cell tolerance to selected viral epitopes should be considered in future vaccine design. During the current grant period, we established an adaptable model system for tolerance that employs anti-mu as a surrogate for antigen. This approach allowed us to examine the ground rules for tolerance (via anergy or apoptosis) in B-cell subsets regardless of specificity, as well as the second messenger pathways which signal unresponsiveness. In this renewal, we will further define the pathways leading to unresponsiveness in unprimed and primed mice, and then utilize this to modulate anti-HIV gp120 humoral responses Using the anti-mu model that we developed, our first goal is to determine the molecular requirements for anergy vs. apoptosis. Normal and Sp6 anti-TNP immunoglobulin transgenic mice will be used to understand the quantitative signals for apoptosis with anti-mu or specific antigen, respectively. The rules for anergy and apoptosis established above will be applied to modulate anti-HIV gp120 peptide responses using highly tolerogenic IgG-peptide conjugates. We will couple C1 and C5 peptides (as model epitopes) to human gamma globulin to modulate B-cell responsiveness of murine B cells to these epitopes, and later use transfection of bone marrow cells to maintain tolerance. Next, we will examine tolerance induction to gp120 epitopes in human CD4 transgenic mice in a model system which allows analysis of T-cell decay in the presence of anti- gpl20 and soluble envelope protein. Ultimately, we will apply the principles above to regulate human B-cell responses in vitro and upon transfer to SCID or RAG- recipients in vivo. These studies will provide insight into mechanisms of B-cell apoptosis and will allow us to manipulate responsiveness of human B-cells to gp120 epitopes, in order to increase the effectiveness of HIV vaccines.